AMPK-independent down-regulation of cFLIP and sensitization to TRAIL-induced apoptosis by AMPK activators

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a TNF superfamily member that is being considered as a new strategy in anticancer therapy because of its ability to induce apoptosis, alone or in combination with other stimuli, in many cancer cells. AMP-activated protein kinase (AMPK) is an evolutionarily conserved key regulator of cellular energy homeostasis that protects the cell from energy depletion and stress by activating several biochemical pathways that lead to the conservation, as well as generation, of ATP. Here we report that a number of AMPK activators, including the small molecule activator A-769662, markedly sensitize TRAIL-resistant breast cancer cells to TRAIL-induced apoptosis. However, silencing AMPKα1 expression with siRNA or over-expression of DN-AMPKα1 does not inhibit AICAR, glucose deprivation, phenformin or A-769662-induced sensitization to TRAIL. Furthermore, the expression of constitutively active AMPK subunits does not sensitize resistant breast cancer cells to TRAIL-induced apoptosis. The cellular FLICE-inhibitory proteins (cFLIP_L and cFLIP_S) were significantly down-regulated following exposure to AMPK activators through an AMPK-independent mechanism. Furthermore, in cells over-expressing cFLIP_L, sensitization to TRAIL by AMPK activators was markedly reduced. In summary, our results indicate that AMPK activators facilitate the activation by TRAIL of an apoptotic cell death program through a mechanism independent of AMPK and dependent on the down-regulation of cFLIP levels.     

Trichostatin A sensitizes human ovarian cancer cells to TRAIL-induced apoptosis by down-regulation of c-FLIPL via inhibition of EGFR pathway

TRAIL-resistant cancer cells can be sensitized to TRAIL by combination therapy. In this study, we investigated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, to overcome the TRAIL resistance in human ovarian cancer cells. Co-treatment of human ovarian cancer cells with TSA and TRAIL synergistically inhibited cell proliferation and induced apoptosis. The combined treatment of ovarian cancer SKOV3 cells with TSA and TRAIL significantly activated caspase-8 and truncated Bid, resulting in the cytosolic accumulation of cytochrome c as well as the activation of caspase-9 and -3. Moreover, we found that down-regulation of c-FLIP_L might contribute to TSA-mediated sensitization to TRAIL-induced apoptosis in SKOV3 cells, and this result was supported by showing that down- or up-regulation of c-FLIP_L with transfection of siRNA or plasmid sensitized or made SKOV3 cells resistant to TRAIL-induced apoptosis, respectively. TSA or co-treatment with TSA alone and TRAIL also resulted in down-regulation of EGFR1/2 and dephosphorylation of its downstream targets, AKT and ERK. Treatment of SKOV3 cells with PKI-166 (EGFR1/2 inhibitor), LY294002 (AKT inhibitor), and PD98059 (ERK inhibitor) decreased c-FLIP_L expression and co-treatment with TRAIL further reduced the level of c-FLIP_L, respectively, as did TSA. Collectively, our data suggest that TSA-mediated sensitization of ovarian cancer cells to TRAIL is closely correlated with down-regulation of c-FLIP_L via inhibition of EGFR pathway, involving caspase-dependent mitochondrial apoptosis, and combination of TSA and TRAIL may be an effective strategy for treating TRAIL-resistant human ovarian cancer cells.     

Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.     

Pentoxifylline augments TRAIL/Apo2L mediated apoptosis in cutaneous T cell lymphoma (HuT-78 and MyLa) by modulating the expression of antiapoptotic proteins and death receptors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising anticancer agent but cutaneous T lymphoma cells (CTCL) are less sensitive to TRAIL-induced apoptosis. Here, we report that pentoxifylline (PTX), a phosphodiesterase inhibitor, augments TRAIL-mediated apoptosis in HuT-78 and MyLa cells through modulating extrinsic death receptors and intrinsic mitochondria dependent pathways. Our results clearly show that PTX augments TRAIL-mediated activation of caspase-8 and induces cleavage of Bid, although PTX alone cannot activate caspase-8. This is followed by cytochrome c release and subsequent, activation of caspase-9 and caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). Combined treatment downregulates the expression of various antiapoptotic proteins including c-FLIP, Bcl-xl, cIAP-1, cIAP-2 and XIAP. PTX induces the expression of death receptors DR4 and DR5 on cell surface of both the cell types where c-Jun NH2-terminal kinase (JNK) pathway plays an important role. Moreover, combined silencing of DR4 and DR5 by small interfering RNA abrogates the ability of PTX to induce TRAIL-mediated apoptosis. Thus, this is the first demonstration that PTX can potentiate TRAIL-mediated apoptosis through downregulation of cell survival gene products and upregulation of death receptors.     

17-Allylamino-17-demethoxygeldanamycin overcomes TRAIL resistance in colon cancer cell lines

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising candidate for treatment of cancer, but displays variable cytotoxicity in cell lines. The mechanisms of sensitivity and resistance have not been fully elucidated; both AKT and NF-kappaB pathways may modulate cytotoxic responses. We have shown that the Hsp90 inhibitor 17-AAG enhances the cytotoxicity of oxaliplatin in colon cancer cell lines through inhibition of NF-kappaB. We analyzed the effects of TRAIL and 17-AAG in combination in a series of nine colon cancer cell lines and characterized activation of the pathways to apoptosis. IC(50) values for a 72 h exposure to TRAIL ranged from 30 to 4000 ng/ml. Cytotoxicity assays demonstrated additivity or synergism of the TRAIL/17-AAG combination in all cell lines, with combination indices at IC(50) ranging from 0.53 to 1. The sensitizing effect of 17-AAG was greater in the TRAIL-resistant cell lines. In TRAIL-resistant cell lines, the combination of 17-AAG and TRAIL resulted in activation of both extrinsic and intrinsic apoptotic pathways, though with quantitative differences between HT29 and RKO cells: differential effects of 17-AAG on AKT and NF-kappaB characterized these cell lines. In both cell lines, the combination also led to down-regulation of X-linked inhibitor of apoptosis protein (XIAP) and enhanced activation of caspase-3. We conclude that either AKT or NF-kappaB may promote resistance to TRAIL in colon cancer cells, and that the ability of 17-AAG to target multiple putative determinants of TRAIL sensitivity warrants their further investigation in combination.     

Anoikis: a necessary death program for anchorage-dependent cells

Cell to matrix adhesion is a key factor for cellular homeostasis and disruption of such interaction has adverse effects on cell survival. It leads to a specific type of apoptosis known as “anoikis” in most non-transformed cell types. This kind of apoptosis following loss of cell anchorage is important for development, tissue homeostasis and several diseases. Integrins sense mechanical forces arising from the matrix, thereby converting these stimuli to downstream signals modulating cell viability. Anchorage-independent growth is a crucial step during tumorigenesis and in particular during the metastatic spreading of cancer cells. The disruption of the tight control leading an “homeless” cell to death is therefore able to violate the cell defences against transformation. This review analyses the recent investigations into the molecular mechanisms governing anoikis, discussing the different ways in which adhesion can influence this process and addressing the relevance of this unique apoptosis mode in the development of metastatic cancers, as well as in other diseases.     

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.     

Guggulsterone sensitizes hepatoma cells to TRAIL-induced apoptosis through the induction of CHOP-dependent DR5: involvement of ROS-dependent ER-stress

Guggulsterone (GGS) has anti-tumor and anti-angiogenesis potential by suppressing nuclear factor-κB and STAT3 activity. Although GGS has been suggested as a potential therapeutic agent for treating various cancers, the underlying molecular mechanisms are unknown. Therefore, we investigated whether GGS sensitizes hepatocellular carcinoma cells (HCC) to apoptosis mediated by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with a GGS/TRAIL combination involved the loss of mitochondrial transmembrane potential and consequent activation of caspases. GGS also induced upregulation of the death receptor DR5 for TRAIL. The effects seemed to be associated with eIF2α and CHOP activation, which are related to the endoplasmic reticulum (ER) stress response and apoptosis. This relationship was suggested by the observation that CHOP downregulation by specific siRNA attenuated both GGS-mediated DR5 upregulation and the cytotoxicity induced by GGS/TRAIL co-treatment. Moreover, salubrinal, a specific eIF-2α phosphorylation-inducing agent, enhanced the expression of CHOP and DR5 induced by GGS and sensitized cells to GGS/TRAIL-induced apoptosis. Thus, GGS-induced eIF2α phosphorylation seems to be important for CHOP and DR5 upregulation. Furthermore, these events were accompanied by an increase in the generation of reactive oxygen species. Pretreatment with N-acetyl-l-cysteine and glutathione inhibited GGS-induced ER-stress, and CHOP and DR5 upregulation and almost completely blocked GGS/TRAIL-induced apoptosis. These results collectively indicate that DR5 induction via eIF-2α and CHOP is crucial for the marked synergistic effects induced by TRAIL and GGS. Taken together, these results indicate that a GGS/TRAIL combination could represent a novel important tool for cancer therapy.     

Role of the Fas/Fas ligand system in female reproductive organs: survival and apoptosis

For centuries, the question of "whether there is life after death" has intrigued the mind of philosophers and the same question fascinates researchers in the field of apoptosis today. The death of a cell is by no means the end of the story. On the contrary, growing evidence suggests that the clearance of apoptotic bodies by macrophages is an important regulatory component in tissue renewal. Without death by apoptosis, the life of reproductive tissues and their function would not be possible. The survival signals that counteract cell death also prepare the cells for apoptosis, and dead cells are important stimuli for tissue survival. The Fas/Fas ligand system is an important mediator of apoptosis and is an excellent example of this apparently contradictory phenomenon.     

Ursolic acid promotes the release of macrophage migration inhibitory factor via ERK2 activation in resting mouse macrophages

Macrophage migration inhibitory factor (MIF) plays some pivotal roles in innate immunity and inflammation. Ursolic acid (UA), an anti-inflammatory triterpene carboxylic acid, was recently reported to induce the release of pro-inflammatory mediators in resting macrophages (Mvarphi). We investigated the effects of UA on MIF protein release in resting RAW264.7 mouse Mvarphi, and found that it decreased intracellular MIF protein levels and promoted the release of MIF into the culture media in dose- and time-dependent manners, without affecting mRNA levels. Further, the triterpene strikingly induced activation of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) within 30min, whereas no phosphorylation of p38 MAPK or JNK protein was observed. In addition, UA-promoted MIF release was significantly inhibited by PD98059, a MEK1/2 inhibitor, while siRNA for ERK2, but not ERK1, significantly decreased the amount of MIF protein released. These results suggest that UA triggers the release of intracellular MIF protein through the ERK2 activation.     

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl⁺ chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl⁺ cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl⁺ cells.     

Induction of apoptosis by cordycepin via reactive oxygen species generation in human leukemia cells

Cordycepin (3′-deoxyadenosin), a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris, one of the top three renowned traditional Chinese medicines. Cordycepin has been shown to possess many pharmacological activities including immunological stimulation, and anti-bacterial, anti-viral, and anti-tumor effects. However, the mechanisms underlying its anti-cancer mechanisms are not yet understood. In this study, the apoptotic effects of cordycepin were investigated in human leukemia cells. Treatment with cordycepin significantly inhibited cell growth in a concentration-dependent manner by inducing apoptosis but not necrosis. This induction was associated with generation of reactive oxygen species (ROS), mitochondrial dysfunction, activation of caspases, and cleavage of poly(ADP-ribose) polymerase protein. However, apoptosis induced by cordycepin was attenuated by caspase inhibitors, indicating an important role for caspases in cordycepin responses. Administration of N-acetyl-l-cysteine, a scavenger of ROS, also significantly inhibited cordycepin-induced apoptosis and activation of caspases. These results support a mechanism whereby cordycepin induces apoptosis of human leukemia cells through a signaling cascade involving a ROS-mediated caspase pathway.     

Regulation of proliferation, survival and apoptosis by members of the TNF superfamily

Tumor necrosis factor (TNF) was first identified in 1984 as a cytokine with anti-tumor effects in vitro and in vivo. Extensive research since then has shown that there are at least 18 distinct members of the TNF super family and they exhibit 15-25% amino acid sequence homology with each other. These family members bind to distinct receptors, which are homologous in their extracellular domain. These cytokines have been implicated in a wide variety of diseases including tumorigenesis, septic shock, viral replication, bone resorption, rheumatoid arthritis, diabetes, and other inflammatory diseases. TNF blockers have been approved for human use in treating some of these conditions in the United States and other countries. Various members of the TNF super family mediate either proliferation, survival, or apoptosis of cells. Although distinct receptors, all members share a common cell signaling pathway that mediates the activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (e.g. c-jun N-terminal kinase). Regulation of cell growth and activation of NF-kappaB and of c-jun N-terminal kinase by the TNF super family is mediated through sequential activation/association of a set of cell signaling proteins named TNF receptor-associated factors, Fas-associated death domain and FADD-like ICE, caspases, receptor-interacting protein, NF-kappaB-inducing kinases, and IkappaBalpha kinases. Both apoptotic and antiapoptotic signals are activated simultaneously by the same cytokine in the same cell. Together these cytokines regulate cell growth/survival/apoptosis in a complex dance of changing partners and overlapping steps.     

Chemoprevention of liver cancer by plant polyphenols

Primary liver cancer or hepatocellular carcinoma (HCC) is one of the most frequent tumors representing the fifth commonest malignancy worldwide and the third cause of mortality from cancer. Currently, the treatments for HCC are not so effective and new strategies are needed for its fight. Chemoprevention, the use of natural or synthetic chemical agents to reverse, suppress or prevent carcinogenesis is considered an important way for confronting HCC. Many of the chemopreventive agents are phytochemicals, namely non-nutritive plant chemicals with protective or disease preventive properties. In this review, we focus on plant polyphenols, one of the most important classes of phytochemicals, their chemopreventive properties against HCC and discuss the molecular mechanisms accounting for this activity.     

Chemoprevention of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (SCC) is responsible for approximately one-sixth of all cancer-related mortality worldwide. This malignancy has a multifactorial etiology involving several environmental, dietary and genetic factors. Since esophageal cancer has often metastasized at the time of diagnosis, current treatment modalities offer poor survival and cure rates. Chemoprevention offers a viable alternative that could well be effective against the disease. Clinical investigations have shown that primary chemoprevention of this disease is feasible if potent inhibitory agents are identified. The Fischer 344 (F-344) rat model of esophageal SCC has been used extensively to investigate the biology of the disease, and to identify chemopreventive agents that could be useful in human trials. Multiple compounds that inhibit tumor initiation by esophageal carcinogens have been identified using this model. These include several isothiocyanates, diallyl sulfide and polyphenolic compounds. These compounds influence the metabolic activation of esophageal carcinogens resulting in reduced genetic (DNA) damage. Recently, a few agents have been shown to inhibit the progression of preneoplastic lesions in the rat esophagus into tumors. These agents include inhibitors of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and c-Jun [a component of activator protein-1 (AP-1)]. Using a food-based approach to cancer prevention, we have shown that freeze-dried berry preparations inhibit both the initiation and promotion/progression stages of esophageal SCC in F-344 rats. These observations have led to a clinical trial in China to evaluate the ability of freeze-dried strawberries to influence the progression of esophageal dysplasia to SCC.